Jan 27, 2021

cell death & disease Abstract Deregulation of chromatin modifiers, including DNA helicases, is emerging as one of the mechanisms underlying the transformation of anaplastic lymphoma kinase negative (ALK−) anaplastic large cell lymphoma (ALCL). We recently identified the DNA-helicase HELLS as central for proficient ALK−ALCL proliferation and progression. Here we assessed in detail its function by performing RNA-sequencing profiling coupled with bioinformatic prediction to identify HELLS targets and transcriptional cooperators. We demonstrated that HELLS, together with the transcription factor YY1, contributes to an appropriate cytokinesis via the transcriptional regulation of genes involved in cleavage furrow regulation. Binding target promoters, HELLS primes YY1 recruitment and transcriptional activation of cytoskeleton genes including the small GTPases RhoA and RhoU and their effector kinase Pak2. Single or multiple knockdowns of these genes reveal that RhoA and RhoU mediate HELLS effects on cell proliferation and cell division of ALK−ALCLs. Collectively, our work demonstrates the transcriptional role of HELLS in orchestrating a complex transcriptional program sustaining neoplastic features of ALK−ALCL. Introduction Anaplastic large cell lymphomas (ALCLs) are a group of neoplasms arising from the transformation of mature T-cell 1 . The presence of chromosomal rearrangements involving the ALK gene stratifies ALCLs in ALK+ and ALK− identifying two distinct diseases with different clinical behavior and prognosis 2 , 3 , 4 . ALK− are known to be the most aggressive subtype of ALCL and the life expectancy of affected patients is significantly reduced by the lack of effective therapies 4 , 5 , 6 . The molecular bases of ALK−ALCLs remain largely unknown as a consequence of the biological complexity of this disease and of its relative rarity that reduces the possibility of extensive profiling 7 , 8 , 9 , 10 , 11 . Understanding the mechanisms that underline the development and evolution of ALK−ALCLs is crucial to define the molecular vulnerabilities of these lymphomas and to develop specific therapeutic strategies. DNA helicases are a class of enzymes whose primary function is to unpack DNA. Considered as molecular motors, these proteins unwind the DNA exploiting ATP hydrolysis, thus facilitating replication and transcription 12 , 13 , 14 . For their importance in DNA maintenance, repair, and chromosomal segregation, helicases are considered guardian of the genomic stability 15 , 16 . Thus, it is not surprising that genetic or transcriptional alterations in many members of this family have been linked to different disease conditions including predisposition to cancer 17 , 18 , 19 , 20 . Besides, the relevance of these enzymes in promoting transcription initiation and cancer progression, has recently started to emerge as essential mechanism to explain their contribution to cell biology 17 , 21 , 22 . In virtue of their centrality in this fundamental mechanism, helicases are currently counted among the most appealing targets for cancer therapies. We recently demonstrated that HELLS, a DNA helicase of the SWI/SNF2 family, is required for proficient ALK−ALCLs proliferation. We showed that HELLS is a downstream target of STAT3 and that its expression is controlled by the ALK−ALCLs specific lncRNA BlackMamba. Besides, we demonstrated that BlackMamba interacts with HELLS driving its positioning on target promoters, suggesting that the role of this helicase in this tumor setting may rely on its transcriptional activity 8 . In this work, we explored in detail the molecular function of HELLS investigating the transcriptional program through which this helicase supports ALK−ALCLs. We provided evidence that HELLS coordinates the expression of a program of genes involved in cytoskeleton organization and cytokinesis thus orchestrating timing of cell division. We also showed that YY1 is a central partner of HELLS in supporting this program. Materials and methods Cell culture and treatments The human ALK−ALCL cell line MAC2A was a kind gift of Dr. Giorgio Inghirami. The human Breast Implanted Associated (BIA)-ALCL cell line TLBR-2 was a kind gift of Dr. Alain Epstein. Cell identity was determined yearly. All cell lines were genotyped and routinely tested for Mycoplasma contamination. Cell lines were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS at 37 °C in an atmosphere of 5% CO2. TLBR-2 cells were supplemented with IL2 (20U/ml). Doxycycline hyclate was purchased from Sigma and dissolved in H2O. Cell growth and cell division For cell growth assays, cells were washed with phosphate-buffered saline, seeded at 2.5×105 cells/ml and treated with 100 nM doxycycline. Viable cells were counted by trypan blue exclusion. Plasmids and viral infections pLKO Tet-On vectors expressing shRNAs against HELLS and lncRNA BlackMamba were generated by cloning synthetic double-stranded oligonucleotides into pLKO Tet-On vector (Addgene #21915). Vectors were packaged into lentiviral particles HEK 293T-cell line and used for infection of low passages MAC2A or TLBR-2 at multiplicity of infection. Cells were selected with 0.5 or 1 μg/ml of puromycin (MAC2A and TLBR-2 respectively) for 3 days. The list of shRNAs sequences is provided in Supplementary Table 1 . siRNA transfection MAC2A and TLBR-2 cells (1×10^6) were transfected with 30 nM siRNA concentration for single KD. SiRNA transfections were performed using the Cell Line Nucleofector Kit SF and Amaxa 4D Nucleofector (program DS-130 for TLBR-2, FI115 for MAC2A). Twenty-four hours after transfection, cells were harvested and plated 2.5 × 105 cells/ml. For siRNA scramble, we used a Silencer Select negative control (Ambion, Life Technologies). For PAK2 and RHOA we used a Silencer Select Validated siRNAs, ID:s10022 and ID:s759, respectively (Ambion, Life Technologies). For RHOU we used two different Silencer Selected Pre-designed siRNAs ID:224502, ID: s33826 (Ambion, Life Technologies). For YY1, we used a Silencer Select Validated siRNAs: ID:s14958 (Ambion, Life Technologies) RNA extraction and quantitative PCR (qRT-PCR) Total RNA was extracted by TRIzol (Thermo Fisher Scientific) according to the manufacture’s instructions. One microgram of total RNA was retrotranscribed using the iScript cDNA kit, (Biorad). The amplified transcript level of each specific gene was normalized on CHMP2A housekeeping. ΔΔCt quantification method was used for RT-qPCR analyses. The list of primers used is provided in Supplementary Table 2 . Western blot Western blot analysis was performed using standard techniques 8 . The primary antibodies were: HELLS (Rabbit mAb#7998, 1:1000 Cell Signaling Technology), γ PAK2 (E-9 Mouse mAb sc-373740, 1:1000 Santa Cruz Biotechnology, Inc), RHOA (26C4 Mouse mAb sc-418, 1:1000 Santa Cruz Biotechnology, Inc), RHOU (Rabbit, PA5-69128, 1:500 Invitrogen), YY1 (Rabbit, D3D4Q, 1:1000, Cell Signaling Technology), β-tubulin antibody (sc-23949, 1:100, Santa Cruz Biotechnology, Inc) and GAPDH (Rabbit mAb#2118, 1:2000, Cell Signaling Technology) All secondary antibodies (rabbit and mouse) were HRP-conjugated (GE Healthcare) and diluted 1:3000. Densitometric analysis was performed using the ImageJ software. Immunofluorescence (IF) Cells were spotted on glass slides using Cytospin (Thermo Scientific), fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 3 min. Dots were blocked in 1% PBS-BSA solution for 40 min at room temperature and incubated with phalloidin (Alexa Fluor® 488, Thermo Fisher) for actin staining for 50 min. Dots were washed in PBS for three times and nuclei were stained with DAPI. For microtubules staining we used β-tubulin antibody (sc-23949, 1:100, Santa Cruz Biotechnology, Inc). Immunofluorescences were detected with Nikon Eclipse (Ni) microscope using 60X. Chromatin immunoprecipitation (ChIP) ChIP was performed as previously described 8 . Chromatin was precipitated with antibodies against HELLS (4ug, Rabbit Polyclonal, orb178580, Biorbyt), YY1 (D3D4Q, 1:100, Cell Signaling Technolgy), or IgG-isotype control (#66362, Cell Signaling Technology). Each qRT-PCR value was normalized over the appropriate input control and reported in graphs as a relative fold on IgG. The list of primers used is provided in Supplementary Table 2 . Co-Immunoprecipitation (Co-IP) Cells were collected, crosslinked with 1% formaldehyde for 10 min, treated with 1.25 M glycine for 5 min and resuspended in Buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5% NP-40) supplemented with protease inhibitor for 8 min in ice. After centrifugation at 3000 rpm for 2 min, the supernatant was collected as the cytoplasmic fraction and used as the quality control of the experiment. The pellet was washed two times with BUFFER B (10 mM HEPES pH7.9, 1.5 mM MgCl2, 10 mM KCl), centrifuged at 3000 rpm for 2 min. Nuclei were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with protease inhibitor and kept for 1 h in rotation at 4°C. Nuclei extracts were sonicated using a Bioruptor® Pico sonicator (Diagenode) and centrifuged at 16,000 g for 10 min. Supernatant was kept and was quantified with Bradford. For each experiment, 4 mg of nuclei extracts was used for immunoprecipitation and 150ɥg was kept as input control. Precoating step was performed using Protein A-Sepharose® CL-4B beads (GE Healthcare, Sigma Aldrich), HELLS antibody (Rabbit Polyclonal, orb178580, Biorbyt). Preclearing step was performed using total nuclear lysate and Protein A-Sepharose beads for aspecific removal for 1 h in rotation at 4°C. After centrifugation at 500 g for 5 min, we combined the beads from precoating and supernatant from preclearing steps and kept in rotation overnight at 4°C. After centrifugation of 500 g for 5 min, the supernatant was discarded and washed four times with TBS 1X (50 mM Tris-HCl pH 7.4, 150 mM NaCl). Laemmli Sample Buffer 4x (Biorad) was added to the immunoprecipitated and samples were boiled for 20 min. Co-IP was detected by western blot using the secondary antibody mouse anti-rabbit IgG HRP conjugate (L27A9) (#5127, 1:2000, Cell Signaling Technology). Library preparation and RNA-sequencing RNA seq libraries were obtained starting from 500 ng of total RNA following Illumina TruSeq Stranded TotalRNA preparation protocol. Sequencing was performed using Illumina NEXSeq high-output cartridge (double-stranded, reads length 75bp-2 ×75). A sequencing depth of at least 60 million reads for each sample was guaranteed. Sequencing quality was assessed using the FastQC v0.11.8 software ( www.bioinformatics.babraham.ac.uk/projects/fastqc/ ), showing on average a Phred score per base >34 in each sample. Raw sequences were then aligned to the human reference transcriptome (GRCh38, Gencode release 30) using STAR version 2.7 23 and gene abundances were estimated with RSEM algorithm (v1.3.1) 24 . Differential expression analysis was performed using DESeq2 R package 25 , considering a False Discovery Rate (FDR) of 10% and excluding genes with low read counts. Heatmap representation and unsupervised hierarchical clustering with a complete linkage method were exploited to graphically depict differentially expressed genes (FRD < 0.1). Significant genes underwent enrichment analysis, performed on Gene Ontology biological processes, KEGG and Reactome pathways databases via enrichR package 26 , using a significance threshold of 0.05 on p-value adjusted by Benjamini–Hochberg correction for multiple testing. Transcriptional factors motif enrichment For transcriptional factor motif search, JASPAR 2020 and PROMO (version 3.0.2) software tools were used. A motif similarity threshold of 80% and a dissimilarity level of 15% were respectively applied for JASPAR 2020 and PROMO prediction results. Statistical analysis Statistical analyses were performed using the GraphPad Prism Software (GraphPad). Statistical significance was determined using Student’s t test. Each experiment was replicated multiple time (>3 up to 6). Results HELLS controls ALK−ALCL proliferation by transcriptionally coordinating a panel of cytoskeleton related genes involved in cytokinesis To get insight into the transcriptional regulation of HELLS, we performed an RNA-sequencing profiling in TLBR-2 cells which represent the ALK−ALCL subtype known as Breast Implanted Associated (BIA)-ALCL 8 , 27 . We generated inducible HELLS knockdown (KD) lines (TLBR-2 HELLSKD) using doxycycline (DOX)-inducible shRNA. HELLS KD was assessed by WB and qRT-PCR (Fig. 1 A, B ) and functionally validated by the reduction in the expression of already described HELLS-downstream targets 28 (Supplementary Fig. 1A ). After DOX induction, the gene expression profile of TLBR-2 HELLSKD cells was analyzed and compared to the one obtained from untreated cells used as control. Transcriptional changes observed after HELLSKD revealed 728 differentially expressed genes. 413 were downregulated and 315 were upregulated upon HELLSKD with a FDR < 0.1 (Fig. 1C ). Gene ontology analysis of HELLS-target genes revealed the enrichment of several categories including cell cycle, DNA damage, histone modification, and chromatin organization. Noticeably, top scoring in this list, there were multiple Rho GTPases and cytoskeleton related categories, including cytoskeleton regulation by Rho GTPases and Rho GTPases signaling (Fig. 1 D, E ). This was particularly interesting since we previously reported that the reduced cellular proliferation displayed by ALK−ALCL upon HELLS loss is associated with defects in cytokinesis and a marked increase in multi-nucleated cells of which Rho-GTPases are major players 8 . Fig. 1: HELLS transcriptionally controls cytokinesis. A Western blot shows HELLS expression in TLBR-2 HELLSKD cells after 48 h of doxycycline (DOX) induction. GAPDH was used as housekeeping gene. B qRT-PCR analysis of HELLS expression in TLBR-2 HELLSKD cells after 48 h of doxycycline induction. The values represent mean ± SEM (n = 3) *p < 0.05; **p < 0.01. C The heatmap depicts hierarchical clustering based on the 728 differentially expressed genes, whose read counts are Z-score normalized. Unsupervised hierarchical clustering was performed between DOX and CTRL samples (as indicated by the colored bar on columns) with a complete linkage method. Color intensity for each gene shows Z-score values ranging from red for upregulation and green for downregulation D Most significant enriched pathways (adjusted p-value<0.05) are represented showing the number of DE genes mapped in each considered pathway. E The heatmap depicts validated significantly downregulated genes. Green color bar shows fold difference on Log2 scale calculated between DOX and CTRL samples. Darker green represents the most downregulated genes. Genes in red were selected for further validations. F Immunofluorescence images of TLBR-2 HELLSKD cells and MAC2A HELLSKD cells after 48 h of doxycycline (DOX) induction. Cells were stained with DAPI, F-actin, and β-tubulin antibodies. The white scale bar represents 10 μm. We noticed that while RhoAKD or Pak2KD did not alter the expression of the others, RhoU KD exerted a significant effect on RhoA expression, suggesting a tight interplay between these two proteins. Morphologically, RhoAKD and RhoUKD led to the increase in the percentage of multi-nucleated cells relative to siRNA scramble. By contrast, no such effect was observed for Pak2 silencing. This phenomenon slightly increased when RhoAKD and RhoUKD were combined. On the contrary, combined RhoUKD/Pak2KD and RhoAKD/Pak2KD did not increase the percentage of multi-nucleated cells relative to RhoUKD and RhoAKD, respectively. Consistently, triple KD resulted in an induction of multinuclei comparable to combined RhoAKD/RhoUKD or RhoAKD (Fig. 5C ). To assess if the multi-nucleated phenotype resulted from cytokinesis failure 44 , we quantified cytokinetic cells by immunofluorescence using F-actin and β-tubulin staining. A significant decrease in the percentage of total cytokinetic cells and a relative increase in abnormal cytokinetic cells were observed in single and combined RhoAKD/RhoUKD compared to siRNA scramble (Fig. 5D ). Immunofluorescences showed that both RhoAKD and RhoUKD affected β-tubulin organization at central spindles or midbody structures. The proper F-actin organization at contractile ring was also dramatically affected resulting in an abnormal formation of cleavage furrow (Fig. 5E ). As expected, combined RhoAKD and RhoUKD resulted in a more pronounced phenotype close to the one obtained with HELLSKD (Fig. 5E ). The analysis of cell proliferation was coherent with these observations. Single silencing of RhoU and Pak2 did not affect significantly cell growth while a significant decrease in cell proliferation was observed in RhoAKD in both cell models. Combined RhoUKD/RhoAKD resulted in a cell proliferation reduction similarly to single RhoAKD whereas combined RhoUKD/Pak2KD enhanced the effects of single RhoUKD and Pak2KD but only in TLBR-2. Notably, triple KD resulted in a significant decrease in cell proliferation, but this reduction was similar to RhoAKD or RhoUKD/Pak2KD (Fig. 5F ). Collectively, these data demonstrated that RhoA and RhoU mediate HELLS effects on cell proliferation and cell division of ALK−ALCLs and that RhoA has a prominent role in this process. Discussion Aberrant expression of epigenetic modifiers fostering the transcriptional program of neoplastic T-cells is emerging as a common feature and potential vulnerability of ALK− ALCLs 45 , 46 . In line with this evidence, here we showed that the DNA-helicase HELLS supports ALK−ALCL proliferation by controlling a gene expression program that is functional for the execution of cytokinesis and therefore for proficient cell division (Fig. 6 ). Fig. 6: Proposed molecular mechanism. Schematic representation of HELLS role in the regulation of ALK−ALCLs cytokinesis-related program (created with BioRender.com). HELLS is a multifunctional protein proved to play, among the others, critical roles in DNA methylation, chromatin packaging, and development of lymphoid tissue 47 . Known also as Lymphoid-specific helicase (Lsh) HELLS is required for normal development and survival of lymphoid and other tissues via chromatin organization 48 49 , promotion of DNA double-strand break repair 50 , 51 and chromatin accessibility modification 52 , 53 . In cancer, HELLS is deregulated in several settings i.e. gliomas 54 , retinoblastoma 55 , 56 , prostate 28 , breast carcinomas 57 , 58 , medulloblastoma 59 , leukemia 60 where it promotes cellular proliferation and stemness. A significant part of HELLS activity in these processes is mediated by its transcriptional function. The way through which HELLS controls gene expression is still partially undefined. Its interaction with epigenetic silencers including G9a 61 and DNMTs 62 as well as with transcriptional factors like E2F3 28 , 56 and c-Myc 54 has been described. Here, we added an additional part of information showing that in the specific context of ALK−ALCL, HELLS interacts and functionally cooperates with the transcription factor YY1. We demonstrate that DNA binding motif for YY1 is enriched within the HELLS binding sites and that these two factors physically interact and co-occupy the same regions at the level of target promoters to ensure transcription of a large set of cytokinesis-related genes. YY1 is an ubiquitously expressed transcriptional factor with a fundamental role in embryogenesis, adult hematopoiesis, differentiation, replication, and cellular proliferation 32 , 63 , 64 . YY1 ensures the proper completion of mitosis and its knockdown leads to defects in cytokinesis and accumulation of multi-nucleated cells 65 . YY1 is often deregulated in hematopoietic malignancies where it controls the survival and growth of neoplastic cells 66 , 67 . Coherently, here we reported that in ALK−ALCL loss of YY1 is associated with accumulation of multi-nucleated cells likely attributable to incomplete cytokinesis. The functional relationship between HELLS and YY1 has never been described before. Interestingly, we showed that the loss of HELLS leads to YY1 displacement from DNA. This seems to indicate that HELLS, by altering chromatin accessibility, works by priming the binding of specific transcription factors to target promoters therefore regulating specific set of context specific genes. By RNA-sequencing profiling we showed that a significant part of the HELLS transcriptional program in ALK−ALCL converges on the regulation of cytoskeleton and cytokinesis. To the best of our knowledge this is the first time that the transcriptional activity of HELLS is linked to these biological processes. Cytoskeleton is the structure responsible for cell shape maintenance and organization. It also confers mechanical support to every cellular process from proliferation and division to cell migration, adhesion, and interaction with the surrounding microenvironment. Of note, recent genetic and molecular profiling studies have unveiled a critical role of cytoskeleton during transformation and progression of T-cells 68 , 69 . Although we still lack of a definitive overall view of how cytoskeleton change during lymphomagenesis, the emerging picture suggests that the cytoskeleton transcends the maintenance of cell morphology and polarity providing a more complex support to T-cells in the response to intrinsic and environmental clues. Among HELLS-downstream effectors, we identified several Rho-GTPases and their related proteins including RhoA, RhoU, and Pak2. Single and combined KDs of these proteins highlight that RhoA and RhoU are key effectors of HELLS program controlling both cell proliferation and cell division. RhoA is a key player in T-cell processes 70 and its deregulation has emerged as a central issue of T-lymphoma biology 68 , 70 , 71 , 72 , 73 . Coherently, our results demonstrate that RhoA is a fundamental effector of HELLS-dependent oncogenic program. At the transcriptional level, RhoA results regulated by HELLS but not by YY1 suggesting a more complex regulation of this Rho-GTPase in this setting. Limited information are available on the regulation of RhoA 74 , and given its centrality, additionally studies are needed to better clarify this point. As RhoU can mediate the effects of WNT and STAT3 signaling pathways in regulating cell morphology, cytoskeletal organization, and proliferation 75 , 76 , our data provide a new layer of complexity demonstrating a new role of RhoU in cytokinesis via the STAT3-BlackMamba-HELLS axis. The cooperative role of Rho-GTPases in the execution of neoplastic program driven by HELLS is in agreement with their fundamental role in the regulation of cell proliferation, cell division, and actin polymerization in cancer 77 . Collectively, our data provide novel insights into the mechanism sustaining the progression of ALK−ALCL via the untapped role of HELLS as transcriptional regulator of cytokinesis. Since HELLS is expressed in many tumors and plays a relevant role in the transcription and genomic stability of cancers, its pharmacological inhibition may represent a promising therapeutic strategy in lymphomas and in other human neoplasms. Data availability Raw data files of RNA-sequencing have been deposited in EMBL-EBI ArrayExpress and are accessible through the accession number E-MTAB-9918. References 1. Swerdlow, S. H. et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 127, 2375–2390 (2016). 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